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Transmembrane of RNA Samples (Siphon Blotting Method)

Experiment Summary

This experiment introduces the procedure of transmembrane (i.e., siphon blotting) of RNA samples, etc.

Main Reagents

20×SSC: 175.3 g NaCl, 88.2 g sodium citrate, DEPC water fixed volume 1000ml, NaOH adjusted pH to 7.0, autoclaved and ready for use.

6×SSC: diluted with 20×SSC.

Main Equipment

UV lamp, 80°C oven, UV crosslinker.

Experimental Steps

The gels were rinsed with distilled water for 2 min after electrophoresis and soaked in 20×SSC for 30 min to remove formaldehyde.
A nitrocellulose membrane of equal size to the gel was cut, moistened with distilled water and placed in 20×SSC.
A flat plate was placed on the square plate, and a filter paper was placed on it. The ends of the filter paper were lapped into the plate and immersed in 20×SSC, and the air bubbles between the filter paper and the plate were driven away with a glass rod.
Place the gel upside down on the platform, bottom side up, cut off the lower right corner, and close the perimeter with plastic wrap to prevent the absorbent paper from touching the edge of the gel and thus the plate from short-circuiting the liquid flow.
Place the nitrocellulose film on the glue to drive out the air bubbles.
Two sheets of filter paper are placed on the membrane and 20 layers of absorbent paper are placed on top of it.
A plate is placed on absorbent paper with a weight of about 500 g on it, with the aim of establishing an upward pathway of liquid from the night pool through the gel to the nitrocellulose membrane to elute the RNA in the gel and allow it to aggregate onto the nitrocellulose membrane.
Overnight film transfer at room temperature for more than 16 hours, during which the paper is changed 2-3 times.
After finishing the transfer film, the gel was observed under UV lamp to determine whether the transfer was complete. And mark the serial number, the position of the addition hole and the position of 28S, 18S, the side of the nitrocellulose membrane and the gel in direct contact is the front side.
Soak the nitrocellulose membrane with 6×SSC for 5 min, blot the filter paper, and dry under vacuum at 80°C oven for 2 h-2.5 h or under UV crosslinker for 5 min. If hybridization experiments are not performed immediately, they are placed in clean filter paper and stored temporarily at room temperature.

* For research use only. Not intended for any clinical use.

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